The thing is, I need to sample the different layers of a sheep cortex at different fetal stages, but I need to do it in an unbiased way, for I have to correlate gene expression from one point of it with the gene expression of the exact same point but at a different fetal age.
Any paper you could suggest for a starting point?
And no, I'm not planning to do an immune approach or in situ hybridization, because for that it'd be easier to use a complete slide. I need to extract many samples from that brain (and some other fetal stages as well), extract the RNA and run a microarray.
How thick is sheep cortex at the various developmental stages you wish to sample?
In rodents, distinguishing laminar boundaries in neocortex is a tricky endeavour - the precise locations of these boundaries depends greatly on what markers (e.g. genetically labeled somata, appearance in bright-field microscopy, soma diameter) you use, but also varies substantially between neocortical areas. I believe the same is generally true across mammals.
A perhaps more useful approach is to specify locations based on normalized fractional depth, i.e. where the pia is at y=0 and the white matter at y=1, and then you can identify the location of your samples (or individual neurons) as e.g. y=0.64, or from a range y=0.56-0.73.
If it is important, you can then also map these locations to "named layers", on the basis of a reference atlas, or by doing a few calibration experiments yourself. In other words, you carefully measure the fractional depth of laminar boundaries using the markers of your choice. Assuming that (as in rodents) these boundaries are fairly consistent from animal to animal, you can use these measurements to later map any sample depths as needed.
Another benefit of this approach is that if you later - after sampling the tissue - find that a different set of laminar boundaries is relevant, you can simply remap your samples as appropriate.
I think the most important factor is to obtain uniform sampling across the full cortical depth, for example to ensure 5 samples for every tenth of the cortical thickness (i.e. 5 samples located between 0.3 and 0.4), for a total of 50 samples. Or at whatever resolution you require.
You can find examples of this coordinate system in a variety of slice electrophysiology papers from Gordon Shepherd's lab (see attached).
Article Top-down laminar organization of the excitatory network in m...
Article Laminar Analysis of Excitatory Local Circuits in Vibrissal M...
Article Reciprocal Interareal Connections to Corticospinal Neurons i...
Benjamin you rock dude =) This is just what I needed. I'll try to pull it on fetal sheep somehow.
I'm glad you found my suggestions helpful, and wish you all the best in your work!
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